In the past decade, the science of genetic engineering has developed rapidly. A variety of processes are known for inserting a heterologous gene into bacteria, whereby the bacteria become capable of efficient expression of the inserted genes. Such processes normally involve the use of plasmids which may be cleaved at one or more selected cleavage sites by restriction endonucleases. Typically, a gene of interest is obtained by cleaving one piece of DNA, and the resulting DNA fragment is mixed with a fragment obtained by cleaving a vector such as a plasmid. The different strands of DNA are then connected (“ligated”) to each other to form a reconstituted plasmid. See, for example, U.S. Pat. Nos. 4,237,224 (Cohen and Boyer, 1980); 4,264,731 (Shine, 1981); 4,273,875 (Manis, 1981); 4,322,499 (Baxter et al, 1982), and 4,336,336 (Silhavy et al, 1982).
A variety of other reference works are available. Some of these works describe the natural process whereby DNA is transcribed into mRNA and mRNA is translated into protein, see, e.g., L. Stryer, Biochemistry, 2nd edition, p 559 et seq. (W. H. Freeman and Co., 1981); A. L. Lehninger, Biochemistry, 2nd edition, p. 853 et seq. (Worth Publ., 1975). Other works describe methods and products of genetic manipulation; see, e.g., T. Maniatis et al, Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Labs, 1982); J. K. Setlow and A. Hollaender, Genetic Engineering, Principles and Methods (Plenum Press, 1979). Hereafter, all references will be cited in abbreviated form; a list of complete citations is included after the Examples.
Most of the genetic engineering work performed to date involves the insertion of genes into various types of cells, primarily bacteria such as E. coli, various other microorganisms such as yeast, and mammalian cells. However, many of the techniques and substances used for genetic engineering of animal cells and microorganisms are not directly applicable to genetic engineering involving plants.
As used herein, the term “plant” refers to a multicellular differentiated organism that is capable of photosynthesis, such as angiosperms and multicellular algae. This does not include microorganisms, such as bacteria, yeast, and fungi. The term “plant cell” includes any cell derived from a plant; this includes undifferentiated tissue such as callus or crown gall tumor, as well as plant seeds, propagules, pollen, or plant embryos.
Ti and Ri Plasmids
The tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens has been proposed for use as a natural vector for introducing foreign genetic information into plant cells (Hernalsteen et al 1980; Rorsch and Schilperoort, 1978). Certain types of A. tumefaciens are capable of infecting a wide variety of plant cells, causing crown gall disease. The process of infection is not fully understood. At least part of the Ti plasmid enters the plant cell. Various metabolic alterations occur, and part of the Ti plasmid is inserted into the genome of the plant (presumably into the chromosomes). The part of the Ti plasmid that enters the plant genome is designated as “transferred DNA” (T-DNA). T-DNA is stably maintained in the plant DNA (Chilton et al, 1977; Yadev et al, 1980; Willmitzer et al, 1980; Otten et al, 1981).
Research by several laboratories has led to the characterization of several structural (i.e., protein coding) genes located in T-DNA (Garfinkel et al 1981; Leemans et al 1982), as well as other DNA sequences which appear to serve various other functions. For example, two sequences referred to as the “left border” and the “right border” appear to delineate T-DNA and may be involved in the process whereby T-DNA is transferred into plant chromosomes (Zambryski et al 1982).
A different species of Agrobacterium, A. rhizogenes, carries a “root-inducing” (Ri) plasmid which is similar to the Ti plasmid. Infection of a plant cell by A. rhizogenes causes hairy root disease. Like the Ti plasmid, a segment of DNA called “T-DNA” (also referred to by some researchers as “R-DNA”) is transferred into the plant genome of an infected cell.
Various other bacteria are also reported to be capable of causing genetic transformation of plant cells, including A. rubi and certain bacteria of the genus Rhizobia which have been treated with a mutagenic agent. Hooykaas et al, at page 156 of Setlow and Holaender, 1979.
As used herein, the term plant tumor inducing plasmid includes any plasmid (1) which is contained in a microorganism, other than a virus, which is capable of causing genetic transformation of one or more types of plants or plant cells, and (2) which contains a segment of DNA which is inserted into a plant genome. This includes Ri plasmids.
As used herein, the ter “T-DNA” refers to a segment of DNA in or from Plant tumor inducing plasmid (1) which has been inserted into the genome of one or more types of plant cells, or (2) which is contained in a segment of DNA that is located between two sequences of bases which are capable of serving as T-DNA borders. As used herein, the terms “T-DNA border” and “border” are determined and applied empirically; these terms shall refer to a sequence of bases which appears at or near the end of a segment of DNA which is transferred from a plant tumor inducing plasmid into a plant genome.
Despite the existing knowledge of T-DNA and plant tumor inducing plasmids, no one prior to this invention has been able to utilize these vectors for the introduction of foreign genes which are expressed in genetically modified plants. A variety of obstacles to such use have been encountered in genetic engineering efforts. Such obstacles include:
1) the large size (approximately 200,000 base pairs) and resulting complexity of plant tumor inducing plasmids preclude the use of standard recombinant DNA techniques to genetically modify and/or insert foreign genes into specific sites in the T-DNA. For example, there are no known unique restriction endonuclease cleavage sites in a Ti plasmid (Leemans et al, 1982).
2) the T-DNA, which is inserted into and expressed in plant cells, contains genes which are involved in the production of high levels of phytohormones in the transformed plant cells (Leemans et al 1982). The high levels of phytohormones interfere with the normal metabolic and regenerative process of the cells, and prevent the formation of phenotypically normal plants from the cells (Braun and Wood, 1976; Yang et al, 1980). Exceptions to this are rare cases where the T-DNA has undergone extensive spontaneous deletions in planta to eliminate those genes involved in phytohormone production. Under these conditions, normal plants are reported to be obtainable at low frequency (Otten et al, 1981). However, the T-DNA genes involved in phytohormone production could not be deleted prior to this invention, since they were very important in the identification and/or selection of transformed plant cells (Marton et al, 1979):
As described above, simple recombinant DNA techniques for introducing foreign genes into plasmids are not applicable to the large Ti plasmid. As a result, several indirect methods have been developed and are discussed below. The first reported use of the Ti plasmid as a vector was in model experiments in which bacterial transposons were inserted into T-DNA and subsequently introduced into plant cells. The bacterial transposons were reported to be stably maintained in the plant genome (Hernalsteens et al, 1980; Garfinkel et al 1981). However, in these cases the transformed tumor tissues were found to be incapable of regeneration into normal plants, and there was no reported evidence for the expression of bacterial genes in the plant cells. In addition, because the insertion of bacterial transposons is believed to be essentially random, a great deal, of effort was required to identify and localize the position of the inserted DNA in these examples. Therefore, this approach is not likely to be useful to introduce desired genes in a predictable manner into plants.
Other researchers have reported the use of intermediate vectors which replicate in both E. coli and A. tumefaciens (Matzke and Chilton, 1981; Leemans et al 1981; Garfinkel et al, 1981). The intermediate vectors contain relatively small subfragments of the Ti plasmid which can be manipulated using standard recombinant DNA techniques. The subfragments can be modified by the deletion of specific sequences or by the insertion of foreign genes at specific sites. The intermediate vectors containing the modified T DNA subfragment are then introduced into A. tumefaciens by transformation or conjugation. Double recombination between the modified T-DNA fragment on the intermediate vector and its wild-type counterpart on the Ti plasmid results in the replacement of the wild-type copy with the modified fragment. Cells which contain the recombined Ti plasmids can be selected using appropriate antibiotics.
Various foreign DNA's have been inserted at specific sites in the T-DNA by this method and they have been reported to be stably transferred into plant cells (Matzke and Chilton, 1981, Leemans et al 1981, 1982). However, such foreign genes have not been reported to be capable o′ f expression in plant cells, and the transformed plant cells remain incapable of regeneration into normal plants. Furthermore, in the procedure described above, it is preferred for a double crossover event to occur, in order to substitute the modified DNA fragment for the wild-type copy. A single crossover results in the formation of a co-integrate plasmid which contains two copies of the T-DNA subfragments. This duplication is undesirable in these methods since homologous recombination, which can occur in A. tumefaciens cells or in plant cells, can result in the loss of the inserted foreign gene(s).
A major disadvantage of the above methods is that the frequency of double recombination is quite low, about 10−4 to 10−9 (Leemans et al, 1981) and it requires extensive effort to identify and isolate the rare double-crossover recombinants. As a result, the number and types of experiments which can be performed using existing methods for genetically engineering the Ti plasmid is severely limited.
Other Means for Inserting DNA into Plant Cells
A variety of other methods have been reported for inserting DNA into plant cells. One such method involves the use of lipid vesicles, also called liposomes, to encapsulate one or more DNA molecules. The liposomes and their DNA contents may be taken up by plant cells; see, e.g., Lurquin, 1981. If the inserted DNA can be incorporated into the plant genome, replicated, and inherited, the plant cells will be transformed.
Other alternate techniques involve contacting plant cells with DNA which is complexed with either (a) polycationic substances, such as poly-L-ornithine (Davey et al, 1980), or (b) calcium phosphate (Krens et al, 1982). Using these techniques, all or part of a Ti plasmid has been reportedly inserted into plant cells, causing tumorigenic alteration of the plant cells.
Another method has been developed involving the fusion of bacteria, which contain desired plasmids, with plant cells. Such methods involve converting the bacteria into spheroplasts and converting the plant cells into protoplasts. Both of these methods remove the cell wall barrier from the bacterial and plant cells, using enzymic digestion. The two cell types can then be used together by exposure to chemical agents, such as polyethylene glycol. See Hasezawa et al, 1981.
However, all of the foregoing techniques suffer from one or more of the following problems:
1. transformation efficiencies reported to date have been very low;
2. only small DNA molecules can be inserted into plant cells;
3. only small numbers of DNA molecules can be inserted into plant cells; and/or,
4. a gene which is inserted into a plant cell will not be stably maintained by the plant cell unless it is incorporated into the genome of the plant cell, i.e., unless the gene is inserted into a chromosome or plasmid that replicates in the plant cell.
For these and possibly other reasons, no one has yet reported expression of a gene inserted into a plant cell by any of the foregoing techniques, except for the tumorigenic transformations noted above.
Prior to this invention, no satisfactory method existed for the creation and identification of genetically transformed plant cells which could be routinely regenerated into morphologically normal plants.